Project 1: In previous studies, we demonstrated that gene-targeted mice lacking the IL-2 gene (IL-2-/- mice) develop various forms of autoimmunity as well as severe colitis "on demand" following parental immunization with 2,4,6-trinitrophenol (TNP)-conjugated keyhole limpet hemocyanin (KLH). Here we show here that this induction of colitis with TNP-KLH is accompanied by a change in the thymoctye population characterized by decreased numbers of double positive (DP; CD4+ CD8+) thymocytes (IL-2 +/+, 45.2 x 106 vs IL-2 -/-, 23.6 x 10/6) and increased numbers of single positive (SP; CD4+ CD8- or CD4-CD8+) thymocytes (IL-2 +/+, 5.3 x 10/6 vs IL-2 -/-, 20.9 x 10/6). In addition, thymocytes from TNP-KLH-immunized IL-2 -/- mice produce more IFN-gamma and less IL-4 than similarly immunized IL-2 +/+ mice. These defects in thymocyte maturation and lymphokine production are IL-2 driven, since they are prevented when immunized IL-2 -/- mice are co-administered anti-IL-12. In parallel studies, we show that IL-2 -/- mice injected with anti-CD3 exhibit decreased cortical apoptosis as determined by thymocyte numbers and detection of apoptotic cells in situ. In addition, we show that colitis-inducing thymocytes are generated in the KLH immunized IL-2 -/- mouse, since IL-2 +/+ mice develop colitis following injection of small numbers of single positive thymocytes from immunized IL-2 -/- mice but not from IL-2 +/+ mice. Taken together, these data indicate that in the TNP-KLH-immunized IL-2-/- mouse thymocyte selection is impaired and thymocyte maturation is abnormally directed by IL-12 toward the generation of mature, activated Th1-type thymocytes that are capable of mediating colitis. Project 2: In previous studies we have shown that IL-2 -/- mice subjected to a single immunization with TNP-KLH or with another TNP-substituted protein (i.p., in adjuvant) exhibit a massive influx of activated Th1 cells into the colonic lamina propria (LP) followed by the development of a severe, chronic colonic inflammation. In the present study we show that whereas LP T cells of TNP-KLH-immunized IL-2-/- mice do not manifest an early (day 2) rise in TGF-beta production LP T cells from IL-2 +/+ exhibit an approximately 8-fold rise in TGF-beta production; in addition, the T cells from IL-2 -/- mice mount a poor IL-4 response. The relation between TGF-beta production in the LP and the development of colitis was further explored in several ways: 1) LP T cells from PBS- or TNP-KLH-immunized IL-2 -/- mice administered anti-CD3 (i.p.) exhibit a significant rise in TGF-beta production but fail to produce IFN-gamma, and such mice do not develop colitis; 2) TNP-KLH-immunized IL-2 -/- mice administered anti-CD3 and coadministered anti-TGF-beta mAb again give rise to IFN-gamma-producing LP cells, and such mice also develop colitis; and 3) TNP-KLH-immunized IL-2+/+ mice administered anti-TGF-beta mAb exhibit pockets of mononuclear cell infiltrates in the LP. In a final series of studies we show that TNP-KLH-immunized IL-2-/- mice administered anti-CD3 manifest T cells in the LP that produce significant amounts of IL-4; however, coadministration of anti-IL-4 mAb does not induce colitis, suggesting that the lack of IL-4 production in TNP-KLH-immunized IL-2-/- mice does not significantly account for the development of colitis. These results indicate that the tendency of IL-2-/- mice to develop chronic colonic inflammation is due to a Th1 cell response in the LP that is not appropriately counterregulated by the production of the suppressor cytokine, TGF-beta. Project 3: In previous studies we performed an extensive analysis of the chronic intestinal inflammation induced in mice by intra-rectal administration of 2,4,6 trinitrobenzene sulfonic acid (TNBS). We showed that this inflammation mimics Crohn's disease and is treatable with anti-IL-12 administration. In the present study we show that both TNBS colitis and the colitis associated with IL-10 deficiency are associated with activation of the transcription factor NF-kappaB component, p65, the transcription factor critical to the production of TNF-alpha and other pro-inflammatory proteins. In addition, we show that local administration of phosphorothioate oligonucleotides down-regulates p65 expression and, in doing so, down-regulates TNF-alpha production. Finally, we show that such administration abrogates clinical and histologic signs of colitis. These data thus provide direct evidence fo the central importance of NF-kappaB in the pathogenesis of chronic intestinal inflammation and suggest a potential therapeutic utility of p65 antisense oligonucleotides as a novel molecular approach for the treatment of patients with Crohn's disease.